The ability of the immune system to generate immense repertoires of clonally distributed antigen receptors on T and B lymphocytes depends on the process of V(D)J recombination. V(D)J recombination occurs in a highly regulated fashion during the early stages of T and B lymphocyte development Cis-acting elements within T cell receptor (TCR) and immunoglobulin loci impart developmental regulation to V(D)J recombination by functioning as region-specific and developmental stage-specific modifiers of chromatin structure. The goal of this proposal is to understand the basic mechanisms underlying this regulation, with a specific focus on the TCR alpha/delta locus. This locus provides a unique model with which to unravel complex regulatory mechanisms because it contains distinct arrays of gene segments that are activated according to a well-defined developmental program. Recent progress has provided insight into factors that direct TCR alpha recombination events. In Specific Aim I genetically altered alleles will be created and analyzed to test roles for Valpha promoters, Jalpha promoters, and the TCRalpha enhancer as regulators of primary and secondary Valpha to Jalpha recombination. By creating and analyzing alleles with introduced transcription terminators, Specific Aim II will address whether germline transcription can regulate chromatin structure, gene segment recombination, and the function of nearby promoters. Gene targeting will be used in Specific Aim III to determine whether V gene segment promoters determine which V gene segments contribute to the adult and fetal TCR delta repertoires. In Specific Aim IV, three dimensional (3D) fluorescence in situ hybridization and chromosome conformation capture will be used to ask whether large scale changes in nuclear position and locus conformation contribute to locus regulation, and whether the TCR alpha enhancer regulates these parameters. Successful completion of these studies should provide information that will allow a more complete understanding of regulation at the TCR alpha/delta locus, and will provide general lessons that will be applicable to the regulation of other antigen receptor loci as well. Ultimately, these studies will help us to understand how the immune system develops the ability to create a repertoire of antigen receptors that are essential for the recognition and control of foreign pathogens, and how it may function to modulate the receptor repertoire in the face of tolerance or autoimmunity.